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Virus vs protein purification

virus protein purification

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#1 Thomson

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Posted 12 March 2018 - 09:59 PM

I am looking to purify virus that is incorporated with a protein (a luminescence tag).

So I collected supernatant of infected cells. The virus containing supernatant I then centrifuged at max speed for 90min and discard the supernatant. The pelleted virion I resuspend in PBS and detect the protein using luminescence substrate.

 

My question is, when I centrifuge as in above condition, can I expect the pelleted is indeed just virion, and any other protein that was expressed by the cells should be in the supernatant after centrifugation. Protein weight much lighter and should not be able to pellet without any precipitation strategy right?



#2 bob1

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Posted 13 March 2018 - 05:29 AM

The pellet will not just be virion - there will be cellular debris in there too. To isolate pure virus you will need to use density gradient isolation.



#3 Thomson

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Posted 26 March 2018 - 05:59 PM

The pellet will not just be virion - there will be cellular debris in there too. To isolate pure virus you will need to use density gradient isolation.

I layered the virus-containing culture supernatant (clarified from low speed centrifugation and 0.45um filter) on top of 20% sucrose and centrifuge 13200rpm for 90min. Discard the supernatant and recover the pelleted virion in PBS.

My issue I faced now is there are still significant cellular protein contaminant in the pelleted virion. Since cellular debris is filtered out by 0.45um, then I supposed these contaminant proteins are in soluble form, why would they be pelleted? Aren't they should be in supernatant?



#4 bob1

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Posted 27 March 2018 - 08:46 AM

Considering that bacteria can easily pass through 0.45 um filters, I would be very surprised if there isn't still cellular debris in the filtrate. However, assuming that your sucrose cushion is the same density as your virion, then you should have a reasonably pure virion preparation. You can perform a couple of these centrifugation steps, one with density greater than or equal that of your virion and one with lower density to help eliminate the remaining contaminants.







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